Cell-density-dependent expression of p35, a lipoprotein of B. burgdorferi has been described by us. Here we further assess this phenomenon by investigating whether other antigens of B. burgdorferi, including some well-characterized ones, are similarly regulated. Low passage spirochetes (strain JD1) were inoculated into fresh BSK-H medium at a concentration of 105 cells/ml and allowed to multiply and reach stationary phase (approximately 108 cells/ml) over an 8-day period. Cells were harvested at various times along the growth curve and processed for Western and Northern blot analyses. To verify the reversibility of the phenomenon of cell-density-dependent expression, cells in stationary phase were again diluted into fresh medium at 105 cells/ml and allowed to undergo another two cycles of growth; during the second cycle cells were sampled in the mid-log phase (log-revertant). The expression of BmpD (P37) and P66, as assessed by Western blot analysis, increased significantly at the onset of stationary phase, as compared to their expression in mid-log phase. Northern blotting revealed a corresponding spike in the steady state level of bmpD transcript at the onset of the stationary phase. Northern blotting is yet to be performed for the p66 mRNA. In contrast, the expression of BmpA (P39), OspA, OspB, OspC, p72, p93 and flagellin all remained unchanged throughout growth of the spirochetes in culture. Western blots developed with serum from monkeys with an acute B. burgdorferi infection revealed two additional antigens, 16 kDa and 26 kDa, whose expression steadily increased during growth in culture. These findings provide additional candidate genes whose expression can be investigated in vitro in an otherwise intractable organism that cycles between arthropod and mammalian hosts.